Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex.

The in vitro adenovirus (Ad) DNA replication system provides an assay to study the interaction of viral and host replication proteins with the DNA template in the formation of the preinitiation complex. This initiation system requires in addition to the origin DNA sequences 1) Ad DNA polymerase (Pol), 2) Ad preterminal protein (pTP), the covalent acceptor for protein-primed DNA replication, and 3) nuclear factor I (NFI), a host cell protein identical to the CCAAT box-binding transcription factor. The interactions of these proteins were studied by coimmunoprecipitation and Ad origin DNA binding assays. The Ad Pol can bind to origin sequences only in the presence of another protein which can be either pTP or NFI. While NFI alone can bind to its origin recognition sequence, pTP does not specifically recognize DNA unless Ad Pol is present. Thus, protein-protein interactions are necessary for the targetting of either Ad Pol or pTP to the preinitiation complex. DNA footprinting demonstrated that the Ad DNA site recognized by the pTP.Pol complex was within the first 18 bases at the end of the template which constitutes the minimal origin of replication. Mutagenesis studies have defined the Ad Pol interaction site on NFI between amino acids 68-150, which overlaps the DNA binding and replication activation domain of this factor. A putative zinc finger on the Ad Pol has been mutated to a product that fails to bind the Ad origin sequences but still interacts with pTP. These results indicate that both protein-protein and protein-DNA interactions mediate specific recognition of the replication origin by Ad DNA polymerase.

Retinal pigment epithelium protein of 65 kDA gene-linked retinal degeneration is not modulated by chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C/ chondroitin sulfate proteoglycan 5.

PURPOSE: To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65⁻/⁻ mouse model of Leber congenital amaurosis. METHODS: We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5⁻/⁻/Rpe65⁻/⁻). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Transcriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6. RESULTS: No differences in the progression of retinal degeneration were observed between the Rpe65⁻/⁻ and Cspg5⁻/⁻/Rpe65⁻/⁻ mice. No retinal phenotype was detected in the late postnatal and adult Cspg5⁻/⁻ mice, when compared to the wild-type mice. CONCLUSIONS: Despite the previously reported upregulation of Cspg5 during retinal degeneration in Rpe65⁻/⁻ mice, no protective effect or any involvement of Cspg5 in disease progression was identified.

Crystallization and preliminary X-ray diffraction studies of Drosophila melanogaster Gαo-subunit of heterotrimeric G protein in complex with the RGS domain of CG5036.

Regulator of G-protein signalling (RGS) proteins negatively regulate heterotrimeric G-protein signalling through their conserved RGS domains. RGS domains act as GTPase-activating proteins, accelerating the GTP hydrolysis rate of the activated form of Gα-subunits. Although omnipresent in eukaryotes, RGS proteins have not been adequately analysed in non-mammalian organisms. The Drosophila melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization of the complex of the two proteins using PEG 4000 as a crystallizing agent and preliminary X-ray crystallographic analysis are reported. Diffraction data were collected to 2.0 Å resolution using a synchrotron-radiation source.

Cross-species analysis of the replication complex of Old World arenaviruses reveals two nucleoprotein sites involved in L protein function.

Lassa virus (LASV) causing hemorrhagic Lassa fever in West Africa, Mopeia virus (MOPV) from East Africa, and lymphocytic choriomeningitis virus (LCMV) are the main representatives of the Old World arenaviruses. Little is known about how the components of the arenavirus replication machinery, i.e., the genome, nucleoprotein (NP), and L protein, interact. In addition, it is unknown whether these components can function across species boundaries. We established minireplicon systems for MOPV and LCMV in analogy to the existing LASV system and exchanged the components among the three systems. The functional and physical integrity of the resulting complexes was tested by reporter gene assay, Northern blotting, and coimmunoprecipitation studies. The minigenomes, NPs, and L proteins of LASV and MOPV could be exchanged without loss of function. LASV and MOPV L protein was also active in conjunction with LCMV NP, while the LCMV L protein required homologous NP for activity. Analysis of LASV/LCMV NP chimeras identified a single LCMV-specific NP residue (Ile-53) and the C terminus of NP (residues 340 to 558) as being essential for LCMV L protein function. The defect of LASV and MOPV NP in supporting transcriptional activity of LCMV L protein was not caused by a defect in physical NP-L protein interaction. In conclusion, components of the replication complex of Old World arenaviruses have the potential to functionally and physically interact across species boundaries. Residue 53 and the C-terminal domain of NP are important for function of L protein during genome replication and transcription.

Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2.

CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis.

Dynamic behavior of Arabidopsis eif4a-iii, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia

Here, we identify the Arabidopsis thaliana ortholog of the mammalian DEAD box helicase, eIF4A-III, the putative anchor protein of exon junction complex (EJC) on mRNA. Arabidopsis eIF4A-III interacts with an ortholog of the core EJC component, ALY/Ref, and colocalizes with other EJC components, such as Mago, Y14, and RNPS1, suggesting a similar function in EJC assembly to animal eIF4A-III. A green fluorescent protein (GFP)-eIF4A-III fusion protein showed localization to several subnuclear domains: to the nucleoplasm during normal growth and to the nucleolus and splicing speckles in response to hypoxia. Treatment with the respiratory inhibitor sodium azide produced an identical response to the hypoxia stress. Treatment with the proteasome inhibitor MG132 led to accumulation of GFP-eIF4A-III mainly in the nucleolus, suggesting that transition of eIF4A-III between subnuclear domains and/or accumulation in nuclear speckles is controlled by proteolysis-labile factors. As revealed by fluorescence recovery after photobleaching analysis, the nucleoplasmic fraction was highly mobile, while the speckles were the least mobile fractions, and the nucleolar fraction had an intermediate mobility. Sequestration of eIF4A-III into nuclear pools with different mobility is likely to reflect the transcriptional and mRNA processing state of the cell.

Analysis of protein-protein interactions in the feline calicivirus replication complex

Caliciviruses are a major cause of gastroenteritis in humans and cause a wide variety of other diseases in animals. Here, the characterization of protein-protein interactions between the individual proteins of Feline calicivirus (FCV), a model system for other members of the family Caliciviridae, is reported. Using the yeast two-hybrid system combined with a number of other approaches, it is demonstrated that the p32 protein (the picornavirus 2B analogue) of FCV interacts with p39 (2C), p30 (3A) and p76 (3CD). The FCV protease/RNA polymerase (ProPol) p76 was found to form homo-oligomers, as well as to interact with VPg and ORF2, the region encoding the major capsid protein VP1. A weak interaction was also observed between p76 and the minor capsid protein encoded by ORF3 (VP2). ORF2 protein was found to interact with VPg, p76 and VP2. The potential roles of the interactions in calicivirus replication are discussed.

GTP binding controls complex formation by the human ROCO protein MASL1

The human ROCO proteins are a family of multi-domain proteins sharing a conserved ROC-COR supra-domain. The family has four members: leu- cine-rich repeat kinase 1 (LRRK1), leucine-rich repeat kinase 2 (LRRK2), death-associated protein kinase 1 (DAPK1) and malignant fibrous histiocy- toma amplified sequences with leucine-rich tandem repeats 1 (MASL1). Previous studies of LRRK1/2 and DAPK1 have shown that the ROC (Ras of complex proteins) domain can bind and hydrolyse GTP, but the cellular consequences of this activity are still unclear. Here, the first biochemical characterization of MASL1 and the impact of GTP binding on MASL1 complex formation are reported. The results demonstrate that MASL1, similar to other ROCO proteins, can bind guanosine nucleotides via its ROC domain. Furthermore, MASL1 exists in two distinct cellular com- plexes associated with heat shock protein 60, and the formation of a low molecular weight pool of MASL1 is modulated by GTP binding. Finally, loss of GTP enhances MASL1 toxicity in cells. Taken together, these data point to a central role for the ROC/GTPase domain of MASL1 in the reg- ulation of its cellular function.

Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and ARP2/3 complex

The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott-Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp(-/-) mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet-platelet interaction and their absence contributes to the bleeding diathesis of Wiskott-Aldrich syndrome.